chapter 11 dna polymorphisms and human identification

Chapter 11Chapter 11

DNA Polymorphisms and Human Identification

ObjectivesObjectives

Compare and contrast different types of polymorphisms.

Define restriction fragment length polymorphisms. Describe short tandem repeat structure and

nomenclature. Describe gender identification using the amelogenin

locus. Illustrate the use of STR for bone marrow engraftment

monitoring. Define single nucleotide polymorphisms. Discuss mitochondrial DNA typing.

PolymorphismPolymorphism

A DNA polymorphism is a sequence difference compared to a reference standard that is present in at least 1–2% of a population.

Polymorphisms can be single bases or thousands of bases.

Polymorphisms may or may not have phenotypic effects.

Polymorphic DNA SequencesPolymorphic DNA Sequences

Polymorphisms are found throughout the genome.

If the location of a polymorphic sequence is known, it can serve as a landmark or marker for locating other genes or genetics regions.

Each polymorphic marker has different versions or alleles.

Types of Polymorphic DNA Types of Polymorphic DNA SequencesSequences

RFLP: restriction fragment length polymorphisms

VNTR: variable number tandem repeats (8 to >50 base pairs)

STR: short tandem repeats (1–8 base pairs)

SNP: single-nucleotide polymorphisms

Restriction Fragment Length Restriction Fragment Length PolymorphismsPolymorphisms

Restriction fragment sizes are altered by changes in or between enzyme recognition sites.

GTCCAGTCTAGCGAATTCGTGGCAAAGGCTCAGGTCAGATCG CTTAAGCACCGTTTCCGA

GTCCAGTCTAGCGAAATCCGTGGCCAAGGCTCAGGTCAGATCG CTTTAGGCACCGGTTCCGA

Point mutationsGTCCAGTCTAGCGAAGCGAATTCGTGGCAAAGGCTCAGGTCAGATCG CTTCGCTTAAGCACCG TTTCCGA

Insertion (duplication)

Fragment insertion (or deletion)

GTTCTAGCGAATTCGTGGCAAA GGCTGAATTCGTGGTCAGATCGCTTAAGCACCGTTTCCGACTTAAGCACC

GTTCTAGCGAATTCGTGGCAAAAAA GGCTGAATTCGTGGTCAGATCG CTTAAGCACCGTTTTT TCCGACTTAAGCACC

Restriction Fragment Length Restriction Fragment Length Polymorphisms Polymorphisms

The presence of RFLP is inferred from changes in fragment sizes.

1 2

A B C

+ -AGATCT ATATCTTCTAGA TATAGA

1 2 Size Number+ + A,B,C 3+ - A, (B+C) 2- + (A+B), C 2- - (A+B+C) 1

+/+ +/- -/+ -/-

Polymorphism

Restriction site

Gel bandpattern

Restriction Fragment Length Restriction Fragment Length Polymorphisms Polymorphisms

The presence of RFLP is inferred from changes in fragment sizes.

Probe

Southern blot band patterns

1 2

A B C

1 2 Fragments visualized+ + B+ - (B+C)- + (A+B)- - (A+B+C)

Genotype Fragments visualizedI ++/+- B, (B+C)II +-/-+ (A+B), (B+C)III ++/-- B,(A+B+C)

+/+ +/- -/+ -/-

I II III

Restriction Fragment Length Restriction Fragment Length Polymorphisms Polymorphisms

RFLP genotypes are inherited. For each locus, one allele is inherited from each

parent. Father Mother Locus Locus 1 2 1 2

Parents

Child

Locus 1 2

Southern blot band patterns

Parentage Testing by RFLPParentage Testing by RFLP

Which alleged father’s genotype has the paternal alleles?

AF 1

AF2 Mother Locus Locus Locus 1 2 1 2 1 2

Child Locus

1 2

Evidence Testing by RFLPEvidence Testing by RFLP

Which suspect—S1 or S2—was at the crime scene? (V = victim, E = crime scene evidence, M = molecular

weight standard) M S1 S2 V E M

M S1 S2 V E M

M S1 S2 V E M

Locus 1 Locus 2 Locus 3

Short Tandem Repeat Short Tandem Repeat Polymorphisms (STR)Polymorphisms (STR)

STR are repeats of nucleotide sequences. AAAAAA… - mononucleotide ATATAT… - dinucleotide TAGTAGTAG… - trinucleotide TAGTTAGTTAGT… - tetranucleotide TAGGCTAGGCTAGGC… - pentanucleotide

Different alleles contain different numbers of repeats. TTCTTCTTCTTC - four repeat allele TTCTTCTTCTTCTTC - five repeat allele

Short Tandem Repeat Short Tandem Repeat PolymorphismsPolymorphisms

STR alleles can be analyzed by fragment size (Southern blot).

GTTCTAGCGGCCGTGGCAGCTAGCTAGCTAGCTGCTGGGCCGTGGCAAGATCGCCGGCACCG TCGATCGATCGATCGACGACCCGGCACC

One repeat unit

tandem repeat

GTTCTAGCGGCCGTGGCAGCTAGCTAGCTGCTGGGCCGTGGCAAGATCGCCGGCACCG TCGATCGATCGACGACCCGGCACC

Restriction site

Allele 1

Allele 2

AlleleM 1 2 M

Short Tandem Repeat Short Tandem Repeat PolymorphismsPolymorphisms

STR alleles can also be analyzed by amplicon size (PCR).

....TCATTCATT CATT CATT CATTCATT CAT....

....AGTAAGTAAGTAAGTAAGTAAGTAAGTA....

....TCAT TCATT CATTCATT CATT CATTCATTCAT....

....AGTAAGTAAGTAAGTAAGTAAGTAAGTAAGTA....

Allele 1

Allele 2

PCR products:Allele 1 187 bp (7 repeats)Allele 2 191 bp (8 repeats)

7/8

(Genotype)

Short Tandem Repeat Short Tandem Repeat PolymorphismsPolymorphisms

Allelic ladders are standards representing all alleles observed in a population.

(Allelic ladder)

11 repeats

5 repeats

Genotype: 7,9 Genotype: 6,8

Short Tandem Repeat Short Tandem Repeat PolymorphismsPolymorphisms

Multiple loci are genotyped in the same reaction using multiplex PCR.

Allelic ladders must not overlap in the same reaction.

Short Tandem Repeat Short Tandem Repeat Polymorphisms by Multiplex PCRPolymorphisms by Multiplex PCR

FGA

TPOX

D8S1179

vWA

PentaE

D18S51

D2S11

THO1

D3S1358

AmelogeninAmelogenin Locus,Locus, HUMAMEL HUMAMEL

The amelogenin locus is not an STR. The HUMAMEL gene codes for amelogenin-like

protein. The gene is located at Xp22.1–22.3 and Y.

X allele = 212 bp Y allele = 218 bp

Females (X, X) - homozygous Males (X, Y) - heterozygous

Analysis of Short Tandem Repeat Analysis of Short Tandem Repeat Polymorphisms by PCRPolymorphisms by PCR

STR genotypes are analyzed using gel or capillary gel electrophoresis.

Genotype: 7,9

11 repeats

5 repeats11 repeats 5 repeats

(Allelic ladder)

STR-PCRSTR-PCR

STR genotypes are inherited.

One allele is inherited from each parent.

Child’s alleles

Mother’s alleles

Father’s alleles

Parentage Testing by STR-PCRParentage Testing by STR-PCR

Which alleged father’s genotype has the paternal alleles?

Locus Child Mother 1 2D3S1358 16/17 16 17 17vWA 14/18 16/18 14/15 16/17FGA 21/24 20/21 24 24TH01 6 6/9.3 6/9 6/7TPOX 10/11 10/11 8/11 8/9CSF1PO 11/12 12 11 11/13D5S818 11/13 10/11 13 9/13D13S317 9/12 9 12/13 11/12

Evidence Testing by STR-PCREvidence Testing by STR-PCR

Which suspect—S1 or S2—was at the crime scene? (V = victim, E = crime scene evidence, AL = Allelic

ladder)

AL S1 S2 V E M

AL S1 S2 V E M

AL S1 S2 V E M

Locus 1 Locus 2 Locus 3

Short Tandem Repeat Short Tandem Repeat Polymorphisms: Y-STRPolymorphisms: Y-STR

The Y chromosome is inherited in a block without recombination.

STR on the Y chromosome are inherited paternally as a haplotype.

Y haplotypes are used for exclusion and paternal lineage analysis.

Chimerism Testing Using STRChimerism Testing Using STR

Allogeneic bone marrow transplants are monitored using STR.

Autologous transplant

Allogeneictransplant

Recipientreceives his or her own purged cells

Recipient receives donor cells

A recipient with donor marrow is a chimera.

Chimerism Testing Using STRChimerism Testing Using STR

There are two parts to chimerism testing: pretransplant informative analysis and post-transplant engraftment analysis

Before transplantDonor Recipient

After transplant

Complete (Full) Mixed Graft failure

Chimerism Testing Using STR:Chimerism Testing Using STR:Informative AnalysisInformative Analysis

STR are scanned to find informative loci (donor alleles differ from recipient alleles).

Which loci are informative?

M D R D R D R D R D R

Locus: 1 2 3 4 5

Chimerism Testing Using STR:Chimerism Testing Using STR:Informative AnalysisInformative Analysis

There are different degrees of informativity.

With the most informative loci, recipient bands or peaks do not overlap stutter in donor bands or peaks. Stutter is a technical artifact of the PCR

reaction in which a minor product of n-1 repeat units is produced.

Donor

Recipient

Recipient

Donor

Stutter

Examples of Informative LociExamples of Informative Loci (Type 5) [Thiede et al., Leukemia 18:248 (2004)](Type 5) [Thiede et al., Leukemia 18:248 (2004)]

Donor

Recipient

Examples of Noninformative Loci Examples of Noninformative Loci (Type 1)(Type 1)

vWA TH01 Amel TPOX CSF1PO

Chimerism Testing Using STR:Chimerism Testing Using STR:Informative AnalysisInformative Analysis

Which loci are informative?

Chimerism Testing Using STR:Chimerism Testing Using STR:Engraftment AnalysisEngraftment Analysis

Using informative loci, peak areas are determined in fluorescence units or from densitometry scans of gel bands.

A(R) = area under recipient-specific peaks A(D) = area under donor-specific peaks

A(D) A(R)

A(R) + A(D)

Chimerism Testing Using STR:Chimerism Testing Using STR:Engraftment AnalysisEngraftment Analysis

Formula for calculation of % recipient or % donor (no shared alleles).

% Recipient DNA = A(R) + A(D)

A(R) × 100

% Donor DNA = A(R) + A(D)

A(D) × 100

Chimerism Testing Using STR:Chimerism Testing Using STR:Engraftment AnalysisEngraftment Analysis

Calculate % recipient DNA in post-1 and post-2:

Use the area under these peaks to calculate percentages.

Chimerism Analysis of Cellular Chimerism Analysis of Cellular SubsetsSubsets

Cell subsets (T cells, granulocytes, NK cells, etc.) engraft with different kinetics.

Analysis of cellular subsets provides a more detailed description of the engrafting cell population.

Analysis of cellular subsets also increases the sensitivity of the engraftment assay.

Chimerism Analysis of Cellular Chimerism Analysis of Cellular SubsetsSubsets

T cells (CD3), NK cells (CD56), granulocytes, myeloid cells (CD13, CD33), myelomonocytic cells (CD14), B cells (CD19), stem cells (CD34)

Methods Flow cytometric sorting Immunomagnetic cell sorting Immunohistochemistry + XY FISH

R D T G

R = Recipient allelesD = Donor allelesT = T-cell subset (mostly recipient)G = Granulocyte subset (mostly donor)

Chimerism Analysis of Cellular Chimerism Analysis of Cellular SubsetsSubsets

Detection of different levels of engraftment in cellular subsets is split chimerism.

Single Nucleotide Polymorphisms Single Nucleotide Polymorphisms (SNP)(SNP)

Single-nucleotide differences between DNA sequences.

One SNP occurs approximately every 1,250 base pairs in human DNA.

SNPs are detected by sequencing, melt curve analysis, or other methods.

99% have no biological effect;60,000 are within genes.

5′ AGTCTG 5′ AG(T/A)CTG 5′ AGACTG

T/T T/A A/A

SNP Detection by SequencingSNP Detection by Sequencing

haplotype~10,000 bp

SNP HaplotypesSNP Haplotypes

SNPs are inherited in blocks or haplotypes.

Applications of SNP AnalysisApplications of SNP Analysis

SNPs can be used for mapping genes, human identification, chimerism analysis, and many other applications.

The Human Haplotype Mapping (HapMap) Project is aimed at identifying SNP haplotypes throughout the human genome.

HV 1(342 bp)

HV 2(268 bp)

Mitochondrial genome16, 600 bp

PL

PH1

PH2

Mitochondrial DNA Mitochondrial DNA PolymorphismsPolymorphisms

Sequence differences in the hypervariable regions (HV) of the mitochondrial genome.

Mitochondrial DNA Mitochondrial DNA PolymorphismsPolymorphisms

Mitochondria are maternally inherited. There are an average of 8.5 base

differences in the mitochondrial HV sequences of unrelated individuals.

All maternal relatives will have the same mitochondrial sequences.

Mitochondrial typing can be used for legal exclusion of individuals or confirmation of maternal lineage.

SummarySummary

Four types of polymorphisms are used for a variety of purposes in the laboratory: RFLP, VNTR, STR, and SNP.

Polymorphisms are used for human identification and parentage testing.

Y-STR haplotypes are paternally inherited. Polymorphisms are used to measure

engraftment after allogeneic bone marrow transplants.

SummarySummary

Single-nucleotide polymorphisms are detected by sequencing, melt curve analysis, or other methods.

SNPs can be used for the same applications as other polymorphisms.

Mitochondrial DNA typing is performed by sequencing the mitochondrial HV regions.

Mitochondrial types are maternally inherited.